CD-R 1 contains Table 4 formatted in tab-delimited ASCII text and the Sequence Listing formatted in plain ASCII text. The file containing Table 4 is entitled Pa41usxcx9c1.TXT, created on Oct. 12, 2001, and is 110 KB in size. The Sequence Listing is entitled Pa41usxcx9c2.TXT, created on Oct. 12, 2001, and is 2,965 KB in size.
CD-R 2 is an exact copy of CD-R 1. CD-R 2 is labeled with Identification No. PA-0041 US, Copy 2.
CD-R 3 contains the Computer Readable Form of the Sequence Listing in compliance with 37 C.F.R. xc2xa71.821(e), and specified by 37 C.F.R. xc2xa71.824. CD-R 3 is labeled with Identification No. PA-0041 US, copy 3.
The disclosure of Tables 1-25 and the Sequence Listing submitted as an electronic document on compact disc as described above are to be part of the permanent USPTO record of this patent application and are hereby expressly incorporated by reference.
The present invention relates to a combination comprising a plurality of cDNAs which are differentially expressed in human C3A liver cell cultures treated with steroids or synthetic steroid analogues and which may be used entirely or in part to detect metabolic and toxicological responses to treatment with steroids and steroid antagonists.
Array technology can provide a simple way to explore the expression of a single polymorphic gene or the expression profile of a large number of related or unrelated genes. When the expression of a single gene is examined, arrays are employed to detect the expression of a specific gene or its variants. When an expression profile is examined, arrays provide a platform for examining which genes are tissue specific, carrying out housekeeping functions, parts of a signaling cascade, or specifically related to a particular genetic predisposition, condition, disease, or disorder.
The potential application of gene expression profiling is particularly relevant to measuring the toxic response to potential therapeutic compounds and of the metabolic response to therapeutic agents. Diseases treated with steroids and disorders caused by the metabolic response to treatment with steroids include adenomatosis, cholestasis, cirrhosis, hemangioma, Henoch-Schonlein purpura, hepatitis, hepatocellular and metastatic carcinomas, idiopathic thrombocytopenic purpura, porphyria, sarcoidosis, and Wilson disease. Response may be measured by comparing both the levels and sequences expressed in tissues from subjects exposed to or treated with steroid compounds such as mifepristone, progesterone, beclomethasone, medroxyprogesterone, budesonide, prednisone, dexamethasone, betamethasone, or danazol with the levels and sequences expressed in normal untreated tissue.
Steroids are a class of lipid-soluble molecules, including cholesterol, bile acids, vitamin D, and hormones, that share a common four-ring structure based on cyclopentanoperhydrophenanthrene and that carry out a wide variety of functions. Cholesterol, for example, is a component of cell membranes that controls membrane fluidity. It is also a precursor for bile acids which solubilize lipids and facilitate absorption in the small intestine during digestion. Vitamin D regulates the absorption of calcium in the small intestine and controls the concentration of calcium in plasma. Steroid hormones, produced by the adrenal cortex, ovaries, and testes, include glucocorticoids, mineralocorticoids, androgens, and estrogens. They control various biological processes by binding to intracellular receptors that regulate transcription of specific genes in the nucleus. Glucocorticoids, for example, increase blood glucose concentrations by regulation of gluconeogenesis in the liver, increase blood concentrations of fatty acids by promoting lipolysis in adipose tissues, modulate sensitivity to catcholamines in the central nervous system, and reduce inflammation. The principal mineralocorticoid, aldosterone, is produced by the adrenal cortex and acts on cells of the distal tubules of the kidney to enhance sodium ion reabsorption. Androgens, produced by the interstitial cells of Leydig in the testis, include the male sex hormone testosterone, which triggers changes at puberty, the production of sperm and maintenance of secondary sexual characteristics. Female sex hormones, estrogen and progesterone, are produced by the ovaries and also by the placenta and adrenal cortex of the fetus during pregnancy. Estrogen regulates female reproductive processes and secondary sexual characteristics. Progesterone regulates changes in the endometrium during the menstrual cycle and pregnancy.
Steroid hormones are widely used for fertility control and in anti-inflammatory treatments for physical injuries and diseases such as arthritis, asthma, and auto-immune disorders. Progesterone, a naturally occurring progestin, is primarily used to treat amenorrhea, abnormal uterine bleeding, or as a contraceptive. Endogenous progesterone is responsible for inducing secretory activity in the endometrium of the estrogen-primed uterus in preparation for the implantation of a fertilized egg and for the maintenance of pregnancy. It is secreted from the corpus luteum in response to luteinizing hormone (LH). The primary contraceptive effect of exogenous progestins involves the suppression of the midcycle surge of LH. At the cellular level, progestins diffuse freely into target cells and bind to the progesterone receptor. Target cells include the female reproductive tract, the mammary gland, the hypothalamus, and the pituitary. Once bound to the receptor, progestins slow the frequency of release of gonadotropin releasing hormone from the hypothalamus and blunt the pre-ovulatory LH surge preventing follicular maturation and ovulation. Progesterone has minimal estrogenic and androgenic activity. Progesterone is metabolized hepatically to pregnanediol and conjugated with glucuronic acid.
Medroxyprogesterone (MAH), also known as 6xcex1-methyl-17-hydroxyprogesterone, is a synthetic progestin with a pharmacological activity about 15 times greater than progesterone. MAH is used for the treatment of renal and endometrial carcinomas, amenorrhea, abnormal uterine bleeding, and endometriosis associated with hormonal imbalance. MAH has a stimulatory effect on respiratory centers and has been used in cases of low blood oxygenation caused by sleep apnea, chronic obstructive pulmonary disease, or hypercapnia.
Mifepristone, also known as RU-486, is an antiprogesterone drug that blocks receptors of progesterone. It counteracts the effects of progesterone, which is needed to sustain pregnancy. Mifepristone induces spontaneous abortion when administered in early pregnancy followed by treatment with the prostaglandin, misoprostol. Further, studies show that mifepristone at a substantially lower dose can be highly effective as a postcoital contraceptive when administered within five days after unprotected intercourse. This is the xe2x80x9cmorning-after pillxe2x80x9d used in case of contraceptive failure or sexual assault. Mifepristone also has potential uses in the treatment of breast and ovarian cancers in cases in which tumors are progesterone-dependent. It interferes with steroid-dependent growth of brain meningiomas, and may be useful in treatment of endometriosis by blocking the estrogen-dependent growth of endometrial tissues. It may also be useful in treatment of uterine fibroid tumors and Cushing""s syndrome. Mifepristone binds to glucocorticoid receptors and interferes with cortisol binding. Mifepristone also may act as an anti-glucocorticoid and be effective for treating conditions where cortisol levels are elevated such as AIDS, anorexia nervosa, ulcers, diabetes, Parkinson""s disease, multiple sclerosis, and Alzheimer""s disease.
Danazol is a synthetic steroid derived from ethinyl testosterone. Danazol indirectly reduces estrogen production by lowering pituitary synthesis of follicle-stimulating hormone and LH. Danazol also binds to sex hormone receptors in target tissues, producing anabolic, antiestrogenic, and weakly androgenic activity. Danazol does not possess any progestogenic activity and does not suppress normal pituitary release of corticotropin or release of cortisol by the adrenal glands. Danazol is used in the treatment of endometriosis to relieve pain and inhibit endometrial cell growth. It is also used to treat fibrocystic breast disease and hereditary angioedema.
Corticosteroids are used to relieve inflammation and to suppress the immune response. They inhibit eosinophil, basophil, and airway epithelial cell function by regulation of cytokines that mediate the inflammatory response. They inhibit leukocyte infiltration at the site of inflammation, interfere in the function of mediators of the inflammatory response, and suppress the humoral immune response. Corticosteroids are used to treat allergies, asthma, arthritis, and skin conditions. Beclomethasone is a synthetic glucocorticoid that is used to treat steroid-dependent asthma, to relieve symptoms associated with allergic or nonallergic (vasomotor) rhinitis, or to prevent recurrent nasal polyps following surgical removal. The anti-inflammatory and vasoconstrictive effects of intranasal beclomethasone are 5000 times greater than those produced by hydrocortisone. Budesonide is a corticosteroid used to control symptoms associated with allergic rhinitis or asthma. Budesonide has high topical anti-inflammatory activity but low systemic activity. Dexamethasone is a synthetic glucocorticoid used in anti-inflammatory or immunosuppressive compositions. It is also used in inhalants to prevent symptoms of asthma. Due to its greater ability to reach the central nervous system, dexamethasone is usually the treatment of choice to control cerebral edema. Dexamethasone is approximately 20-30 times more potent than hydrocortisone and 5-7 times more potent than prednisone. Prednisone is metabolized in the liver to its active form, prednisolone, a glucocorticoid with anti-inflammatory properties. Prednisone is approximately 4 times more potent than hydrocortisone and the duration of action of prednisone is intermediate between hydrocortisone and dexamethasone. Prednisone is used to treat allograft rejection, asthma, systemic lupus erythematosus, arthritis, ulcerative colitis, and other inflammatory conditions. Betamethasone is a synthetic glucocorticoid with antiinflammatory and immunosuppressive activity and is used to treat psoriasis and fungal infections such as athlete""s foot and ringworm.
The anti-inflammatory actions of corticosteroids are thought to involve phospholipase A2 inhibitory proteins, collectively called lipocortins. Lipocortins, in turn, control the biosynthesis of potent mediators of inflammation such as prostaglandins and leukotrienes by inhibiting the release of the precursor molecule arachidonic acid. Proposed mechanisms of action include decreased IgE synthesis, increased number of xcex2-adrenergic receptors on leukocytes, and decreased arachidonic acid metabolism. During an immediate allergic reaction such as in chronic bronchial asthma, allergens bridge the IgE antibodies on the surface of mast cells and trigger these cells to release chemotactic substances. Mast cell influx and activation is partially responsible for the inflammation and hyperirritability of the oral mucosa in asthmatic patients. This inflammation can be retarded by administration of corticosteroids.
The effects of steroids on liver metabolism and hormone clearance mechanisms are important to understand the pharmacodynamics of a drug. The human C3A cell line is a clonal derivative of HepG2/C3 (hepatoma cell line, isolated from a 15-year-old male with liver tumor), which was selected for strong contact inhibition of growth. The use of a clonal population enhances the reproducibility of the cells. C3A cells have many characteristics of primary human hepatocytes in culture including i) expression of insulin receptor and insulin-like growth factor II receptor; ii) secretion of a high ratio of serum albumin compared with xcex1-fetoprotein iii) conversion of ammonia to urea and glutamine; iv) metabolism of aromatic amino acids; and v) proliferation in glucose-free and insulin-free medium. The C3A cell line is well established as an in vitro model of the mature human liver (Mickelson et al. (1995) Hepatology 22:866-875; Nagendra et al. (1997) Am J Physiol 272:G408-G416).
The present invention provides a combination comprising a plurality of cDNAs for use in detecting changes in expression of genes encoding proteins that are associated with steroid treatment. Such a combination satisfies a need in the art by providing a set of differentially expressed genes which may be used entirely or in part to detect metabolic and toxicological responses to steroid compounds and therapeutics.
The present invention provides a combination comprising a plurality of cDNAs and their complements which are differentially expressed in human C3A cells treated with steroid compounds and which are selected from SEQ ID NOs:1, 2, 3, 5, 6, 8, 9, 11-14, 16-18, 20, 21, 23-34, 36, 38-40, 42-50, 52-63, 65, 66, 68-71, 73-75, 77-84, 86-111, 113-115, 117-126, 128-142, 145-160, 162-164, 166, 167, 169-172, 174-176, 178-186, 188-197, 199-202, 204-211, 213, 215, 217-228, 230-246, 248-251, 253-259, 261-266, 268-277, 279, 281, 283, 284, 286, 288-292, 294, 295, 297-300, 302-308, 310, 311, 313, 315-320, 322, 323, 325, 327-346, 348-350, 352-357, 359-368, 370-372, 374, 376, 378, 380-389, 391-400, 402, 403, 405-413, 415, 416, 418, 420, 422-426, 428, 429, 431-446, 448, 450-453, 455, 457, 459, 461, 463-466, 468-470, 472-478, 480-488, 490-492, 494-502, 504-506, 508-520, 522-526, 528-530, 532, 534, 535, 537-561, 563-565, 567, 569-575, 577, 579-581, 583, 585-589, 591-614, 616, 618, 619, 621, 623, 625, 627, 629-641, 643, 645-654, 656-663, 665-680, 682-698, 700-708, 710, 712-720, 722-725, 727-730, 732, 734, 735, 737-741, 743-754, 756, 758-768, 770-780, 782-786, 788-792, 794, 796-802, 804-806, 808, 809, 811-814, 816, 817, 819-829, 831, 833, 835, 836, 838-840, 842-888, 890-892, 894-908, 910, 912, 914-927, 929, 930, 932-937, 939-944, 946, 948-957, 959-964, 966-976, 978-988, 990-999, 1001-1008, 1010-1028, 1030-1034, 1036-1085, 1087-1096, 1098-1143 as presented in the Sequence Listing. The level of each cDNA is downregulated or upregulated at least 2.5-fold. In one aspect, the combination is useful to diagnose a liver disorder selected from adenomatosis, cholestasis, cirrhosis, hemangioma, Henoch-Schonlein purpura, hepatitis, hepatocellular and metastatic carcinomas, idiopathic thrombocytopenic purpura, porphyria, sarcoidosis, and Wilson disease and for detecting metabolic and toxicological responses to treatment with steroids. In another aspect, the combination is immobilized on a substrate.
The invention also provides a high throughput method to detect differential expression of one or more of the cDNAs of the combination. The method comprises hybridizing the substrate comprising the combination with the nucleic acids of a sample, thereby forming one or more hybridization complexes, detecting the hybridization complexes, and comparing the hybridization complexes with those of a standard, wherein differences in the size and signal intensity of each hybridization complex indicates differential expression of nucleic acids in the sample. In one aspect, the sample is from a subject with a liver disorder associated with steroid therapy such as adenomatosis, cholestasis, cirrhosis, hemangioma, Henoch-Schonlein purpura, hepatitis, hepatocellular and metastatic carcinomas, idiopathic thrombocytopenic purpura, porphyria, sarcoidosis, and Wilson disease and differential expression determines an early, mid, and late stage of that disorder.
The invention further provides a high throughput method of screening a library or a plurality of molecules or compounds to identify a ligand. The method comprises combining the substrate comprising the combination with a library or plurality of molecules or compounds under conditions to allow specific binding and detecting specific binding, thereby identifying a ligand. The library or plurality of molecules or compounds are selected from DNA molecules, RNA molecules, peptide nucleic acid molecules, mimetics, peptides, transcription factors, repressors, and other regulatory proteins. The invention additionally provides a method for purifying a ligand, the method comprising combining a cDNA of the invention with a sample under conditions which allow specific binding, recovering the bound cDNA, and separating the cDNA from the ligand, thereby obtaining purified ligand.
The invention still further provides an isolated cDNA selected from SEQ ID NOs:2, 17, 23, 24, 31, 32, 54, 57, 58, 60, 69, 73, 81-83, 86, 88, 90, 94-98, 102-105, 108, 113, 120-124, 129, 130, 133-135, 137, 140, 142, 145, 146, 148, 152, 154, 156, 157, 166, 174, 182, 185, 188, 189, 192, 194, 199, 205, 206, 208, 221, 234, 235-239, 253, 256, 258, 261, 271, 290, 297, 304, 307, 331, 338, 342, 343, 352, 354, 355, 360, 362, 364, 365, 380, 381, 395, 396, 402, 410, 412, 424, 428, 432, 435, 436, 438, 440, 444, 450, 464, 465, 472, 476, 477, 480, 487-490, 499-501, 510-512, 515, 519, 524, 528, 529, 538-542, 544, 545, 547, 549, 550, 554, 556, 558, 559, 563, 564, 570-572, 574, 580, 585, 588, 591, 594, 599-607, 648, 649, 651-653, 657, 658, 669, 670, 675, 677, 678, 684, 685, 688, 692-696, 700-705, 716, 728, 729, 739, 743, 748, 749, 751, 761, 763, 766, 767, 772, 783, 785, 790, 797, 798, 800, 805, 812, 813, 821, 825, 828, 839, 843, 846, 851-854, 857-859, 863-867, 873, 875, 879, 881-884, 887, 896, 901, 902, 905, 914, 916, 919, 923, 929, 933, 935, 936, 942, 948, 949-956, 959, 962, 963, 966, 969, 970, 972, 980, 984, 990-993, 998, 1003, 1004, 1007, 1013, 1016, 1020, 1026, 1027, 1030, 1037, 1038, 1040, 1042, 1046, 1051, 1056, 1059, 1063, 1068-1070, 1075, 1078, 1081, 1087, 1088, 1090, 1092-1095, 1099, 1105, 1107, 1108, 1113, 1117, 1118, 1121, 1128, 1129, 1132, 1140, 1141, and 1143 as presented in the Sequence Listing. The invention also provides a vector comprising the cDNA, a host cell comprising the vector, and a method for producing a protein comprising culturing the host cell under conditions for the expression of a protein and recovering the protein from the host cell culture.
The present invention provides a purified protein encoded and produced by a cDNA of the invention. The invention also provides a high-throughput method for using a protein to screen a library or a plurality of molecules or compounds to identify a ligand. The method comprises combining the protein or a portion thereof with the library or plurality of molecules or compounds under conditions to allow specific binding and detecting specific binding, thereby identifying a ligand which specifically binds the protein. The library or plurality of molecules or compounds is selected from aptamers, DNA molecules, RNA molecules, peptide nucleic acid molecules, mimetics, peptides, proteins, agonists, antagonists, antibodies or their fragments, immunoglobulins, inhibitors, drug compounds, and pharmaceutical agents. The invention further provides for using a protein to purify a ligand. The method comprises combining the protein or a portion thereof with a sample under conditions to allow specific binding, recovering the bound protein, and separating the protein from the ligand, thereby obtaining purified ligand. The invention still further provides a composition comprising the protein and a pharmaceutical carrier.
The invention also provides methods for using a protein to prepare and purify polyclonal and monoclonal antibodies which specifically bind the protein. The method for preparing a polyclonal antibody comprises immunizing a animal with protein under conditions to elicit an antibody response, isolating animal antibodies, attaching the protein to a substrate, contacting the substrate with isolated antibodies under conditions to allow specific binding to the protein dissociating the antibodies from the protein, thereby obtaining purified polyclonal antibodies. The method for preparing and purifying monoclonal antibodies comprises immunizing a animal with a protein under conditions to elicit an antibody response, isolating antibody producing cells from the animal, fusing the antibody producing cells with immortalized cells in culture to form monoclonal antibody producing hybridoma cells, culturing the hybridoma cells, and isolating from culture monoclonal antibodies which specifically bind the protein.
The invention provides a purified antibody that specifically binds a protein expressed in a liver disorder. The invention also provides a method for using an antibody to detect expression of a protein in a sample comprising combining the antibody with a sample under conditions which allow the formation of antibody:protein complexes and detecting complex formation, wherein complex formation indicates expression of the protein in the sample.
A portion of the disclosure of this patent document contains material that is subject to copyright protection. The copyright owner has no objection to the facsimile reproduction by anyone of the patent document or the patent disclosure, as it appears in the Patent and Trademark Office patent file or records, but otherwise reserves all copyright rights whatsoever.
The Sequence Listing is a compilation of cDNAs obtained by sequencing and extending clone inserts. Each sequence is identified by a sequence identification number (SEQ ID NO) and by the Incyte identification number (Incyte ID) from which it was obtained.
Table 1 lists the functional annotation of the cDNAs of the present invention. Columns 1 and 2 show the SEQ ID NO and TEMPLATE ID, respectively. Columns 3, 4, and 5 show the GenBank ID, probability score (E-value), and functional annotation, respectively, as determined by BLAST analysis (version 1.4 using default parameters; Altschul (1993) J Mol Evol 36: 290-300; Altschul et al. (1990) J Mol Biol 215:403-410) of the cDNA against GenBank (release 117; National Center for Biotechnology Information (NCBI), Bethesda Md.).
Table 2 shows Pfam annotations of the cDNAs of the present invention. Columns 1 and 2 show the SEQ ID NO and Clone ID, respectively. Columns 3, 4, and 5 show the first residue (START), last residue (STOP), and reading frame, respectively, for the segment of the cDNA identified by Pfam analysis. Columns 6 and 7 show the Pfam description and E-values, respectively, corresponding to the polypeptide domain encoded by the cDNA segment.
Table 3 shows the region of each cDNA encompassed by the clone present on a microarray and identified as differentially expressed. Columns 1 and 2 show the SEQ ID NO and TEMPLATE ID, respectively. Column 3 shows the CLONE ID and columns 4 and 5 show the first residue (START) and last residue (STOP) encompassed by the clone on the template sequence.
Table 4 shows signal peptide and transmembrane regions predicted within the cDNAs of the present invention. Columns 1 and 2 show the SEQ ID NO and TEMPLATE ID, respectively. Columns 3, 4, and 5 show the first residue (START), last residue (STOP), and reading frame (FRAME), respectively, for a segment of the cDNA, and column 6 (HIT TYPE) identifies the polypeptide encoded by the segment as either a signal peptide (SP) or transmembrane (TM) domain.
Table 5 lists the tissue distribution of the nucleotide templates. Columns 1 and 2 list the SEQ ID NO and TEMPLATE ID, respectively. Column 3 lists the predominant tissue distribution (TISSUE DISTRIBUTION) as a percentage of total tissues in the LIFESEQ GOLD database (Incyte Genomics, Palo Alto Calif.).
Tables 6-14 show the differentially expressed genes associated with treatment of human C3A cells with various steroid compounds. Differentially expressed genes associated with mifepristone (mife) treatment are shown in Table 6; with progesterone (Prog) treatment in Table 7; with beclomethasone (Beclo) treatment in Table 8; with medroxyprogesterone (MAH) treatment in Table 9; with budesonide (bude) treatment in Table 10; with prednisone (Prdsne) treatment in Table 11; with dexamethasone (Dex) treatment in Table 12; with betamethasone (Betam) treatment in Table 13; and with danazol treatment in Table 14. In each table, genes are grouped by similarity in response to the compound as determined by K-means clustering algorithms; cluster numbers are associated with specific GEM designs.
In Tables 6-14, column 1 shows the Incyte clone ID number; column 2 shows the average log2 differential expression value for treated C3A cells, column 3 shows the range of log2 mean differential expression values for treated C3A cells, columns 4 through 12 show the log2 mean differential expression values for C3A cells treated with the steroid compound at concentrations of 1, 10, and 100 xcexcM for 1, 3, and 6 hours; column 13 shows the cluster number to which the gene was assigned, and column 14 shows the GEM microarray (Incyte Genomics) designation for the gene.
Table 15 lists the genes that showed differential expression with all of the steroid compounds. Column 1 shows the Incyte clone ID number, columns 2 and 3 show the GenBank ID and functional annotation respectively, column 4 shows the average log2 differential expression value for treated C3A cells, and column 5 shows the steroid used in treatment of human C3A liver cells.
Table 16 lists the genes that showed differential expression selectively with only one of the steroid compounds. Column 1 shows the Incyte clone ID number, column 2 shows the average log2 differential expression value for treated C3A cells, and column 3 shows the steroid used in treatment of human C3A liver cells.
Tables 17-25 show groups of genes that are regulated selectively by two or three of the steroids. Table 17 shows groups of genes that are differentially expressed selectively in response to progesterone and either MAH or betamethasone. Table 18 shows groups of genes that are differentially expressed selectively in response to mifepristone and either MAH or danazol. Table 19 shows groups of genes that are differentially expressed selectively in response to MAH and each of the other steroids. Table 20 shows groups of genes that are differentially expressed selectively in response to danazol and mifepristone, MAH or betamethasone. Table 21 shows groups of genes that are differentially expressed selectively in response to dexamethasone and either other corticosteroids or MAH. Table 22 shows groups of genes that are differentially expressed selectively in response to betamethasone and either other corticosteroids, or MAH, progesterone, or danazol. Table 23 shows groups of genes that are differentially expressed selectively in response to beclomethasone and either other corticosteroids or MAH. Table 24 shows groups of genes that are differentially expressed selectively in response to budesonide and either other corticosteroids or MAH. Table 25 shows groups of genes that are differentially expressed selectively in response to prednisone and either other corticosteroids or MAH.
In Tables 17-25, column 1 shows the Incyte clone ID number, column 2 shows the average log2 differential expression value for treated C3A cells, and column 3 shows the steroid used in treatment of human C3A liver cells.
Definitions
xe2x80x9cArrayxe2x80x9d refers to an ordered arrangement of at least two cDNAs on a substrate. At least one of the cDNAs represents a control or standard sequence, and the other, a cDNA of diagnostic interest. The arrangement of from about two to about 40,000 cDNAs on the substrate assures that the size and signal intensity of each labeled hybridization complex formed between a cDNA and a sample nucleic acid is individually distinguishable.
The xe2x80x9ccomplementxe2x80x9d of a nucleic acid molecule of the Sequence Listing refers to a nucleotide sequence which is completely complementary over the full length of the sequence and which will hybridize to the nucleic acid molecule under conditions of high stringency.
A xe2x80x9ccombinationxe2x80x9d comprises at least two and up to 1143 sequences selected from the group consisting of SEQ ID NOs:1-1143 as presented in the Sequence Listing.
xe2x80x9ccDNAxe2x80x9d refers to a chain of nucleotides, an isolated polynucleotide, nucleic acid molecule, or any fragment or complement thereof. It may have originated recombinantly or synthetically, be double-stranded or single-stranded, coding and/or noncoding, an exon with or without an intron from a genomic DNA molecule, and purified or combined with carbohydrate, lipids, protein or inorganic elements or substances. Preferably, the cDNA is from about 400 to about 10000 nucleotides.
The phrase xe2x80x9ccDNA encoding a proteinxe2x80x9d refers to a nucleic acid sequence that closely aligns with sequences which encode conserved regions, motifs or domains that were identified by employing analyses well known in the art. These analyses include BLAST (Basic Local Alignment Search Tool; Altschul (1993) J Mol Evol 36: 290-300; Altschul et al. (1990) J Mol Biol 215-410) which provides identity within the conserved region. Brenner et al. (1998; Proc Natl Acad Sci 95:6073-6078) who analyzed BLAST for its ability to identify structural homologs by sequence identity found 30% identity is a reliable threshold for sequence alignments of at least 150 residues and 40% is a reasonable threshold for alignments of at least 70 residues (Brenner et al., page 6076, column 2).
xe2x80x9cDerivativexe2x80x9d refers to a cDNA or a protein that has been subjected to a chemical modification. Derivatization of a cDNA can involve substitution of a nontraditional base such as queosine or of an analog such as hypoxanthine. These substitutions are well known in the art. Derivatization of a protein involves the replacement of a hydrogen by an acetyl, acyl, alkyl, amino, formyl, or morpholino group. Derivative molecules retain the biological activities of the naturally occurring molecules but may confer advantages such as longer lifespan or enhanced activity.
xe2x80x9cDifferential expressionxe2x80x9d refers to an increased or upregulated or a decreased or downregulated expression as detected by absence, presence, or at least 2.5-fold change in the amount of transcribed messenger RNA or translated protein in a sample.
xe2x80x9cDisorderxe2x80x9d refers to conditions, diseases or syndromes such as adenomatosis, cholestasis, cirrhosis, hemangioma, Henoch-Schonlein purpura, hepatitis, hepatocellular and metastatic carcinomas, idiopathic thrombocytopenic purpura, porphyria, sarcoidosis, Wilson disease, and toxicological responses associated with the use of steroid compounds.
xe2x80x9cFragmentxe2x80x9d refers to a chain of consecutive nucleotides from about 200 to about 700 base pairs in length. Fragments may be used in PCR or hybridization technologies to identify related nucleic acid molecules and in binding assays to screen for a ligand. Nucleic acids and their ligands identified in this manner are useful as therapeutics to regulate replication, transcription or translation.
A xe2x80x9chybridization complexxe2x80x9d is formed between a cDNA and a nucleic acid of a sample when the purines of one molecule hydrogen bond with the pyrimidines of the complementary molecule, e.g., 5xe2x80x2-A-G-T-C-3xe2x80x2 base pairs with 3xe2x80x2-T-C-A-G-5xe2x80x2. The degree of complementarity and the use of nucleotide analogs affect the efficiency and stringency of hybridization reactions.
xe2x80x9cIdentityxe2x80x9d as applied to sequences, refers to the quantification (usually percentage) of nucleotide or residue matches between at least two sequences aligned using a standardized algorithm such as Smith-Waterman alignment (Smith and Waterman (1981) J Mol Biol 147:195-197), CLUSTALW (Thompson et al. (1994) Nucleic Acids Res 22:4673-4680), or BLAST2 (Altschul et al. (1997) supra). BLAST2 may be used in a standardized and reproducible way to insert gaps in one of the sequences in order to optimize alignment and to achieve a more meaningful comparison between them. xe2x80x9cSimilarityxe2x80x9d as applied to proteins uses the same algorithms but takes into account conservative substitutions of nucleotides or residues.
xe2x80x9cLigandxe2x80x9d refers to any agent, molecule, or compound which will bind specifically to a complementary site on a cDNA molecule or polynucleotide, or to an epitope or a protein. Such ligands stabilize or modulate the activity of polynucleotides or proteins and may be composed of inorganic or organic substances including nucleic acids, proteins, carbohydrates, fats, and lipids.
xe2x80x9cOligonucleotidexe2x80x9d refers a single stranded molecule from about 18 to about 60 nucleotides in length which may be used in hybridization or amplification technologies or in regulation of replication, transcription or translation. Substantially equivalent terms are amplimer, primer, and oligomer.
xe2x80x9cPortionxe2x80x9d refers to any part of a protein used for any purpose which retains at least one biological or antigenic characteristic of a native protein; but especially, to an epitope for the screening of ligands or for the production of antibodies.
xe2x80x9cPost-translational modificationxe2x80x9d of a protein can involve lipidation, glycosylation, phosphorylation, acetylation, racemization, proteolytic cleavage, and the like. These processes may occur synthetically or biochemically. Biochemical modifications will vary by cellular location, cell type, pH, enzymatic milieu, and the like.
xe2x80x9cProbexe2x80x9d refers to a cDNA that hybridizes to at least one nucleic acid molecule in a sample. Where targets are single stranded, probes are complementary single strands. Probes can be labeled with reporter molecules for use in hybridization reactions including Southern, northern, in situ, dot blot, array, and like technologies or in screening assays.
xe2x80x9cProteinxe2x80x9d refers to a polypeptide or any portion thereof. An xe2x80x9coligopeptidexe2x80x9d is an amino acid sequence from about five residues to about 15 residues that is used as part of a fusion protein to produce an antibody.
xe2x80x9cPurifiedxe2x80x9d refers to any molecule or compound that is separated from its natural environment and is preferably about 60% free and more preferably about 90% free from other components with which it is naturally associated.
xe2x80x9cSamplexe2x80x9d is used in its broadest sense as containing nucleic acids, proteins, antibodies, and the like. A sample may comprise a bodily fluid; the soluble fraction of a cell preparation, or an aliquot of media in which cells were grown; a chromosome, an organelle, or membrane isolated or extracted from a cell; genomic DNA, RNA, or cDNA in solution or bound to a substrate; a cell; a tissue or a tissue biopsy; a tissue print; a fingerprint, buccal cells, skin, or hair; and the like.
xe2x80x9cSpecific bindingxe2x80x9d refers to a special and precise interaction between two molecules which is dependent upon their structure, particularly their molecular side groups. For example, the intercalation of a regulatory protein into the major groove of a DNA molecule, the hydrogen bonding along the backbone between two single stranded nucleic acids, or the binding between an epitope of a protein and an agonist, antagonist, or antibody.
xe2x80x9cSubstratexe2x80x9d refers to any rigid or semi-rigid support to which cDNAs or proteins are bound and includes membranes, filters, chips, slides, wafers, fibers, magnetic or nonmagnetic beads, gels, capillaries or other tubing, plates, polymers, and microparticles with a variety of surface forms including wells, trenches, pins, channels and pores.
xe2x80x9cVariantxe2x80x9d refers to molecules that are recognized variations of a cDNA or a protein encoded by the cDNA. Splice variants may be determined by BLAST score, wherein the score is at least 100, and most preferably at least 400. Allelic variants have a high percent identity to the cDNAs and may differ by about three bases per hundred bases. xe2x80x9cSingle nucleotide polymorphismxe2x80x9d (SNP) refers to a change in a single base as a result of a substitution, insertion or deletion. The change may be conservative (purine for purine) or non-conservative (purine to pyrimidine) and may or may not result in a change in an encoded amino acid.
The Invention
The present invention provides for a combination comprising a plurality of cDNAs or their complements, SEQ ID NOs:1, 2, 3, 5, 6, 8, 9, 11-14, 16-18, 20, 21, 23-34, 36, 38-40, 42-50, 52-63, 65, 66, 68-71, 73-75, 77-84, 86-111, 113-115, 117-126, 128-142, 145-160, 162-164, 166, 167, 169-172, 174-176, 178-186, 188-197, 199-202, 204-211, 213, 215, 217-228, 230-246, 248-251, 253-259, 261-266, 268-277, 279, 281, 283, 284, 286, 288-292, 294, 295, 297-300, 302-308, 310, 311, 313, 315-320, 322, 323, 325, 327-346, 348-350, 352-357, 359-368, 370-372, 374, 376, 378, 380-389, 391-400, 402, 403, 405-413, 415, 416, 418, 420, 422-426, 428, 429, 431-446, 448, 450-453, 455, 457, 459, 461, 463-466, 468-470, 472-478, 480-488, 490-492, 494-502, 504-506, 508-520, 522-526, 528-530, 532, 534, 535, 537-561, 563-565, 567, 569-575, 577, 579-581, 583, 585-589, 591-614, 616, 618, 619, 621, 623, 625, 627, 629-641, 643, 645-654, 656-663, 665-680, 682-698, 700-708, 710, 712-720, 722-725, 727-730, 732, 734, 735, 737-741, 743-754, 756, 758-768, 770-780, 782-786, 788-792, 794, 796-802, 804-806, 808, 809, 811-814, 816, 817, 819-829, 831, 833, 835, 836, 838-840, 842-888, 890-892, 894-908, 910, 912, 914-927, 929, 930, 932-937, 939-944, 946, 948-957, 959-964, 966-976, 978-988, 990-999, 1001-1008, 1010-1028, 1030-1034, 1036-1085, 1087-1096, 1098-1143 which may be used on a substrate to detect metabolic and toxicological responses to treatment with steroids and steroid antagonists and as diagnostics for liver disorders associated with steroid therapy such as adenomatosis, cholestasis, cirrhosis, hemangioma, Henoch-Schonlein purpura, hepatitis, hepatocellular and metastatic carcinomas, idiopathic thrombocytopenic purpura, porphyria, sarcoidosis, and Wilson disease. These cDNAs represent known and novel genes differentially expressed in human C3A liver cells treated with mifepristone, progesterone, beclomethasone, medroxyprogesterone, budesonide, prednisone, dexamethasone, betamethasone, or danazol. The combination may be used in its entirety or in part, as subsets of downregulated cDNAs or of upregulated cDNAs. SEQ ID NOs:2, 17, 23, 24, 31, 32, 54, 57, 58, 60, 69, 73, 81-83, 86, 88, 90, 94-98, 102-105, 108, 113, 120, 121, 123, 124, 129, 130, 133-135, 137, 140, 142, 145, 146, 148, 152, 154, 156, 157, 166, 174, 182, 185, 188, 189, 192, 194, 199, 205, 206, 208, 221, 234-239, 253, 256, 258, 261, 271, 290, 297, 304, 307, 331, 338, 342, 343, 352, 354, 355, 360, 362, 364, 365, 380, 381, 395, 396, 402, 410, 412, 424, 428, 432, 435, 436, 438, 440, 444, 450, 464, 465, 472, 476, 477, 480, 487-490, 499, 500, 501, 510-512, 515, 519, 524, 528, 529, 538-542, 544, 545, 547, 549, 550, 554, 556, 558, 559, 563, 564, 570-572, 574, 580, 585, 588, 591, 594, 599, 600-607, 648, 649, 651-653, 657, 658, 669, 670, 675, 677, 678, 684, 685, 688, 692-696, 700-705, 716, 728, 729, 739, 743, 748, 749, 751, 761, 763, 766, 767, 772, 783, 785, 790, 797, 798, 800, 805, 812, 813, 821, 825, 828, 839, 843, 846, 851-854, 857-859, 863-867, 873, 875, 879, 881-884, 887, 896, 901, 902, 905, 914, 916, 919, 923, 929, 933, 935, 936, 942, 948, 949, 952-956, 959, 962, 963, 966, 969, 970, 972, 980, 984, 990-993, 998, 1003, 1004, 1007, 1013, 1016, 1020, 1026, 1027, 1030, 1037, 1038, 1040, 1042, 1046, 1051, 1056, 1059, 1063, 1068-1070, 1075, 1078, 1081, 1087, 1088, 1090, 1092-1095, 1099, 1105, 1107, 1108, 1113, 1117, 1118, 1121, 1128, 1129, 1132, 1140, 1141, and 1143 represent novel cDNAs associated with the response of C3A cells to treatment with steroids. Since the novel cDNAs were identified solely by their differential expression, it is not essential to know a priori the name, structure, or function of the gene or the encoded protein. The usefulness of the novel cDNAs exists in their immediate value as diagnostics for liver disorders associated with steroid therapy such as adenomatosis, cholestasis, cirrhosis, hemangioma, Henoch-Schonlein purpura, hepatitis, hepatocellular and metastatic carcinomas, idiopathic thrombocytopenic purpura, porphyria, sarcoidosis, and Wilson disease, and for detection of metabolic and toxicological responses to treatment with steroids and steroid antagonists.
The invention also provides isolated proteins, SEQ ID NOs:4, 7, 10, 15, 19, 22, 35, 37, 41, 51, 64, 67, 72, 76, 85, 112, 116, 127, 143, 144, 161, 165, 168, 173, 177, 187, 198, 203, 212, 214, 216, 229, 247, 252, 260, 267, 278, 280, 282, 285, 287, 293, 296, 301, 309, 312, 314, 321, 324, 326, 347, 351, 358, 369, 373, 375, 377, 379, 390, 401, 404, 414, 417, 419, 421, 427, 430, 443, 447, 449, 454, 456, 458, 460, 462, 467, 471, 479, 489, 493, 503, 507, 521, 527, 531, 533, 536, 562, 566, 568, 576, 578, 582, 584, 590, 615, 617, 620, 622, 624, 626, 628, 642, 644, 655, 664, 681, 699, 709, 711, 721, 726, 731, 733, 736, 742, 755, 757, 769, 781, 787, 793, 795, 803, 807, 810, 815, 818, 830, 832, 834, 837, 841, 889, 893, 909, 911, 913, 928, 931, 938, 945, 947, 958, 965, 977, 989, 1000, 1009, 1029, 1035, 1086, and 1097 which are encoded by the cDNAs of SEQ ID NOs:3, 6, 9, 14, 18, 21, 34, 36, 40, 50, 63, 66, 71, 75, 84, 111, 115, 126, 143, 160, 164, 167, 172, 176, 186, 197, 202, 211, 213, 215, 228, 246, 251, 259, 266, 277, 279, 281, 284, 286, 292, 295, 300, 308, 311, 313, 320, 323, 325, 346, 350, 357, 368, 372, 374, 376, 378, 389, 400, 403, 413, 416, 418, 420, 426, 429, 442, 446, 448, 453, 455, 457, 459, 461, 466, 470, 478, 488, 492, 502, 506, 520, 526, 530, 532, 535, 561, 565, 567, 575, 577, 581, 583, 589, 614, 616, 619, 621, 623, 625, 627, 641, 643, 654, 663, 680, 698, 708, 710, 720, 725, 730, 732, 735, 741, 754, 756, 768, 780, 786, 792, 794, 802, 806, 809, 814, 817, 829, 831, 833, 836, 840, 888, 892, 908, 910, 912, 927, 930, 937, 944, 946, 957, 964, 976, 988, 999, 1008, 1028, 1034, 1085, and 1096 respectively as shown in Table 1.
Agglomerative cluster analysis was used to identify response patterns and to establish relationships between different gene expression profiles. Each gene measurement was normalized by dividing the expression ratios by the maximum value for each time series. The clustering process defined a hierarchical tree with the number of branches intersecting at each branch level of the tree equal to the number of clusters at that level.
Table 1 shows those genes on the array having differential expression (2.5-fold or greater increase or decrease) in treated human C3A liver cell cultures, the encoded protein sequences which correspond to the upregulated or downregulated clones present on the array, and the functional annotation of the template cDNAs as determined by BLAST analysis. Table 2 shows the functional annotation as determined by Pfam analysis. Table 3 shows the positional information of the clone present on the array relative to the nucleotide template sequence. Table 4 shows the functional annotation as determined by Hidden Markov Model analysis for signal peptide (SP) or for transmembrane (TM) regions. Table 5 shows the tissue distribution of the nucleotide template sequences. Tables 6-14 show those genes on the array having differential expression (2.5-fold or greater increase or decrease) in human C3A cells treated with mifepristone, progesterone, beclomethasone, medroxyprogesterone, budesonide, prednisone, dexamethasone, betamethasone, or danazol respectively. Table 15 lists the genes, identified by Incyte Clone ID, that showed differential expression with all of the steroid compounds. Genes represented by Incyte clones 279898 and 4287327 on the microarray are upregulated by all of the steroid compounds. Genes represented by Incyte clones 560115, 1619292, 2769888, 3120209, 3135460, 4408943, 5047895, 5166479, and 5427710 on the microarray are downregulated by all of the steroid compounds. Table 16 lists the genes that showed differential expression selectively with only one of the steroid compounds. Table 17 shows the group of genes that are differentially expressed selectively in response to progesterone and are regulated similarly by either the synthetic progestin MAH or the glucocorticoid betamethasone. Table 18 shows the group of genes that are differentially expressed selectively in response to mifepristone and are regulated similarly by other steroids which bind to sex hormone receptors including MAH or danazol. Table 19 shows the group of genes that are differentially expressed selectively in response to MAH. The synthetic progestin MAH alters the expression of a wider range of genes than progesterone. Table 20 shows the group of genes that are differentially expressed selectively in response to danazol and mifepristone, MAH or betamethasone. Danazol, mifepristone, and MAH show similar effects on differential expression. Danazol and betamethasone show opposing effects on expression. Table 21 shows the group of genes that are differentially expressed selectively in response to dexamethasone and are regulated similarly by either other corticosteroids or MAH. Beclomethasone, compared with the other corticosteroids, regulates the most genes in common with dexamethasone and shows the most similarity in its effects on gene expression. Table 22 shows the group of genes that are differentially expressed selectively in response to betamethasone and are regulated similarly by other corticosteroids, MAH, progesterone, or danazol. Budesonide, compared with the other corticosteroids, regulates the most genes in common with betamethasone and shows the most similarity in its effects on gene expression. Betamethasone in contrast to the other corticosteroids, regulates a significant number of genes in common with progesterone and other steroids which bind sex hormone receptors. Table 23 shows the group of genes that are differentially expressed selectively in response to beclomethasone and are regulated similarly by either other corticosteroids or MAH. Table 24 shows that group of genes that are differentially expressed selectively in response to budesonide and are regulated similarly by either other corticosteroids or MAH. Betamethasone, compared with the other corticosteroids, regulates the most genes in common with budesonide. Table 25 shows the group of genes that are differentially expressed selectively in response to prednisone and are regulated similarly by either other corticosteroids or MAH.
The cDNAs of the invention define a differential expression pattern against which to compare the expression pattern of biopsied and/or in vitro treated human liver tissues. Experimentally, differential expression of the cDNAs can be evaluated by methods including, but not limited to, differential display by spatial immobilization or by gel electrophoresis, genome mismatch scanning, representational discriminant analysis, clustering, transcript imaging and array technologies. These methods may be used alone or in combination.
The combination may be arranged on a substrate and hybridized with tissues from subjects with diagnosed liver disorders associated with steroid therapy such as adenomatosis, cholestasis, cirrhosis, hemangioma, Henoch-Schonlein purpura, hepatitis, hepatocellular and metastatic carcinomas, idiopathic thrombocytopenic purpura, porphyria, sarcoidosis, and Wilson disease to identify those sequences which are differentially expressed and for detection of metabolic and toxicological responses to treatment with steroids and steroid antagonists. This allows identification of those sequences of highest diagnostic and potential therapeutic value. In one embodiment, an additional set of cDNAs, such as cDNAs encoding signaling molecules, are arranged on the substrate with the combination. Such combinations may be useful in the elucidation of pathways which are affected in particular liver disorders associated with steroid therapy or to identify new, coexpressed, candidate, toxicological markers.
In another embodiment, the combination can be used for large scale genetic or gene expression analysis of a large number of novel, nucleic acid molecules. These samples are prepared by methods well known in the art and are from mammalian cells or tissues which are in a certain stage of development; have been treated with a known molecule or compound, such as a cytokine, growth factor, a drug, and the like; or have been extracted or biopsied from a mammal with a known or unknown condition, disorder, or disease before or after treatment. The sample nucleic acid molecules are hybridized to the combination for the purpose of defining a novel gene profile associated with that developmental stage, treatment, or disorder.
cDNAs and Their Uses
cDNAs can be prepared by a variety of synthetic or enzymatic methods well known in the art. cDNAs can be synthesized, in whole or in part, using chemical methods well known in the art (Caruthers et al. (1980) Nucleic Acids Symp Ser (7)215-233). Alternatively, cDNAs can be produced enzymatically or recombinantly, by in vitro or in vivo transcription.
Nucleotide analogs can be incorporated into cDNAs by methods well known in the art. The only requirement is that the incorporated analog must base pair with native purines or pyrimidines. For example, 2,6-diaminopurine can substitute for adenine and form stronger bonds with thymidine than those between adenine and thymidine. A weaker pair is formed when hypoxanthine is substituted for guanine and base pairs with cytosine. Additionally, cDNAs can include nucleotides that have been derivatized chemically or enzymatically.
cDNAs can be synthesized on a substrate. Synthesis on the surface of a substrate may be accomplished using a chemical coupling procedure and a piezoelectric printing apparatus as described by Baldeschweiler et at. (PCT publication WO95/251116). Alternatively, the cDNAs can be synthesized on a substrate surface using a self-addressable electronic device that controls when reagents are added as described by Heller et al. (U.S. Pat. No. 5,605,662). cDNAs can be synthesized directly on a substrate by sequentially dispensing reagents for their synthesis on the substrate surface or by dispensing preformed DNA fragments to the substrate surface. Typical dispensers include a micropipette delivering solution to the substrate with a robotic system to control the position of the micropipette with respect to the substrate. There can be a multiplicity of dispensers so that reagents can be delivered to the reaction regions efficiently.
cDNAs can be immobilized on a substrate by covalent means such as by chemical bonding procedures or UV irradiation. In one method, a cDNA is bound to a glass surface which has been modified to contain epoxide or aldehyde groups. In another method, a cDNA is placed on a polylysine coated surface and UV cross-linked to it as described by Shalon et al. (WO95/35505). In yet another method, a cDNA is actively transported from a solution to a given position on a substrate by electrical means (Heller, supra). cDNAs do not have to be directly bound to the substrate, but rather can be bound to the substrate through a linker group. The linker groups are typically about 6 to 50 atoms long to provide exposure of the attached cDNA. Preferred linker groups include ethylene glycol oligomers, diamines, diacids and the like. Reactive groups on the substrate surface react with a terminal group of the linker to bind the linker to the substrate. The other terminus of the linker is then bound to the cDNA. Alternatively, polynucleotides, plasmids or cells can be arranged on a filter. In the latter case, cells are lysed, proteins and cellular components degraded, and the DNA is coupled to the filter by UV cross-linking.
The cDNAs may be used for a variety of purposes. For example, the combination of the invention may be used on an array. The array, in turn, can be used in high-throughput methods for detecting a related polynucleotide in a sample, screening a plurality of molecules or compounds to identify a ligand, diagnosing liver disorders associated with steroid therapy such as adenomatosis, cholestasis, cirrhosis, hemangioma, Henoch-Schonlein purpura, hepatitis, hepatocellular and metastatic carcinomas, idiopathic thrombocytopenic purpura, porphyria, sarcoidosis, and Wilson disease, detecting metabolic and toxicological responses to treatment with steroids and steroid antagonists, or inhibiting or inactivating a therapeutically relevant gene related to the cDNA.
When the cDNAs of the invention are employed on a microarray, the cDNAs are arranged in an ordered fashion so that each cDNA is present at a specified location. Because the cDNAs are at specified locations on the substrate, the hybridization patterns and intensities, which together create a unique expression profile, can be interpreted in terms of expression levels of particular genes and can be correlated with a particular metabolic process, condition, disorder, disease, stage of disease, or treatment.
Hybridization
The cDNAs or fragments or complements thereof may be used in various hybridization technologies. The cDNAs may be labeled using a variety of reporter molecules by either PCR, recombinant, or enzymatic techniques. For example, a commercially available vector containing the cDNA is transcribed in the presence of an appropriate polymerase, such as T7 or SP6 polymerase, and at least one labeled nucleotide. Commercial kits are available for labeling and cleanup of such cDNAs. Radioactive (Amersham Pharmacia Biotech (APB), Piscataway N.J.), fluorescent (Operon Technologies, Alameda Calif.), and chemiluminescent labeling (Promega, Madison Wis.) are well known in the art.
A cDNA may represent the complete coding region of an mRNA or be designed or derived from unique regions of the mRNA or genomic molecule, an intron, a 3xe2x80x2 untranslated region, or from a conserved motif. The cDNA is at least 18 contiguous nucleotides in length and is usually single stranded. Such a cDNA may be used under hybridization conditions that allow binding only to an identical sequence, a naturally occurring molecule encoding the same protein, or an allelic variant. Discovery of related human and mammalian sequences may also be accomplished using a pool of degenerate cDNAs and appropriate hybridization conditions. Generally, a cDNA for use in Southern or northern hybridizations may be from about 400 to about 6000 nucleotides long. Such cDNAs have high binding specificity in solution-based or substrate-based hybridizations. An oligonucleotide, a fragment of the cDNA, may be used to detect a polynucleotide in a sample using PCR.
The stringency of hybridization is determined by G+C content of the cDNA, salt concentration, and temperature. In particular, stringency is increased by reducing the concentration of salt or raising the hybridization temperature. In solutions used for some membrane based hybridizations, addition of an organic solvent such as formamide allows the reaction to occur at a lower temperature. Hybridization may be performed with buffers, such as 5xc3x97saline sodium citrate (SSC) with 1% sodium dodecyl sulfate (SDS) at 60xc2x0 C., that permit the formation of a hybridization complex between nucleic acid sequences that contain some mismatches. Subsequent washes are performed with buffers such as 0.2xc3x97SSC with 0.1% SDS at either 45xc2x0 C. (medium stringency) or 65xc2x0-68xc2x0 C. (high stringency). At high stringency, hybridization complexes will remain stable only where the nucleic acid molecules are completely complementary. In some membrane-based hybridizations, preferably 35% or most preferably 50%, formamide may be added to the hybridization solution to reduce the temperature at which hybridization is performed. Background signals may be reduced by the use of detergents such as Sarkosyl or Triton X-100 (Sigma Aldrich, St. Louis Mo.) and a blocking agent such as denatured salmon sperm DNA. Selection of components and conditions for hybridization are well known to those skilled in the art and are reviewed in Ausubel et al. (1997, Short Protocols in Molecular Biology, John Wiley and Sons, New York N.Y., Units 2.8-2.11, 3.18-3.19 and 4.6-4.9).
Dot-blot, slot-blot, low density and high density arrays are prepared and analyzed using methods known in the art. cDNAs from about 18 consecutive nucleotides to about 5000 consecutive nucleotides in length are contemplated by the invention and used in array technologies. The preferred number of cDNAs on an array is at least about 100,000, a more preferred number is at least about 40,000, an even more preferred number is at least about 10,000, and a most preferred number is at least about 600 to about 800. The array may be used to monitor the expression level of large numbers of genes simultaneously and to identify genetic variants, mutations, and SNPs. Such information may be used to determine gene function; to understand the genetic basis of a disorder; to diagnose a disorder; and to develop and monitor the activities of therapeutic agents being used to control or cure a disorder. (See, e.g., U.S. Pat. No. 5,474,796; WO95/11995; WO95/35505; U.S. Pat. Nos. 5,605,662; and 5,958,342.)
Screening and Purification Assays
A cDNA may be used to screen a library or a plurality of molecules or compounds for a ligand which specifically binds the cDNA. Ligands may be DNA molecules, RNA molecules, peptide nucleic acid molecules, peptides, proteins such as transcription factors, promoters, enhancers, repressors, and other proteins that regulate replication, transcription, or translation of the polynucleotide in the biological system. The assay involves combining the cDNA or a fragment thereof with the molecules or compounds under conditions that allow specific binding and detecting the bound cDNA to identify at least one ligand that specifically binds the cDNA.
In one embodiment, the cDNA may be incubated with a library of isolated and purified molecules or compounds and binding activity determined by methods such as a gel-retardation assay (U.S. Pat. No. 6,010,849) or a reticulocyte lysate transcriptional assay. In another embodiment, the cDNA may be incubated with nuclear extracts from biopsied and/or cultured cells and tissues. Specific binding between the cDNA and a molecule or compound in the nuclear extract is initially determined by gel shift assay. Protein binding may be confirmed by raising antibodies against the protein and adding the antibodies to the gel-retardation assay where specific binding will cause a supershift in the assay.
In another embodiment, the cDNA may be used to purify a molecule or compound using affinity chromatography methods well known in the art. In one embodiment, the cDNA is chemically reacted with cyanogen bromide groups on a polymeric resin or gel. Then a sample is passed over and reacts with or binds to the cDNA. The molecule or compound which is bound to the cDNA may be released from the cDNA by increasing the salt concentration of the flow-through medium and collected.
The cDNA may be used to purify a ligand from a sample. A method for using a cDNA to purify a ligand would involve combining the cDNA or a fragment thereof with a sample under conditions to allow specific binding, recovering the bound cDNA, and using an appropriate agent to separate the cDNA from the purified ligand.
Protein Production and Uses
The full length cDNAs or fragment thereof may be used to produce purified proteins using recombinant DNA technologies described herein and taught in Ausubel et al. (sunra; Units 16.1-16.62). One of the advantages of producing proteins by these procedures is the ability to obtain highly-enriched sources of the proteins thereby simplifying purification procedures.
The proteins may contain amino acid substitutions, deletions or insertions made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues involved. Such substitutions may be conservative in nature when the substituted residue has structural or chemical properties similar to the original residue (e.g., replacement of leucine with isoleucine or valine) or they may be nonconservative when the replacement residue is radically different (e.g., a glycine replaced by a tryptophan). Computer programs included in LASERGENE software (DNASTAR, Madison Wis.), MACVECTOR software (Genetics Computer Group, Madison Wis.) and RasMol software (Roger Sayles, University of Massachusetts, Amherst Mass.) may be used to help determine which and how many amino acid residues in a particular portion of the protein may be substituted, inserted, or deleted without abolishing biological or immunological activity.
Expression of Encoded Proteins
Expression of a particular cDNA may be accomplished by cloning the cDNA into a vector and transforming this vector into a host cell. The cloning vector used for the construction of cDNA libraries in the LIFESEQ databases may also be used for expression. Such vectors usually contain a promoter and a polylinker useful for cloning, priming, and transcription. An exemplary vector may also contain the promoter for xcex2-galactosidase, an amino-terminal methionine and the subsequent seven amino acid residues of xcex2-galactosidase. The vector may be transformed into competent E. coli cells. Induction of the isolated bacterial strain with isopropylthiogalactoside (IPTG) using standard methods will produce a fusion protein that contains an N terminal methionine, the first seven residues of xcex2-galactosidase, about 15 residues of linker, and the protein encoded by the cDNA.
The cDNA may be shuttled into other vectors known to be useful for expression of protein in specific hosts. Oligonucleotides containing cloning sites and fragments of DNA sufficient to hybridize to stretches at both ends of the cDNA may be chemically synthesized by standard methods. These primers may then be used to amplify the desired fragments by PCR. The fragments may be digested with appropriate restriction enzymes under standard conditions and isolated using gel electrophoresis. Alternatively, similar fragments are produced by digestion of the cDNA with appropriate restriction enzymes and filled in with chemically synthesized oligonucleotides. Fragments of the coding sequence from more than one gene may be ligated together and expressed.
Signal sequences that dictate secretion of soluble proteins are particularly desirable as component parts of a recombinant sequence. For example, a chimeric protein may be expressed that includes one or more additional purification-facilitating domains. Such domains include, but are not limited to, metal-chelating domains that allow purification on immobilized metals, protein A domains that allow purification on immobilized immunoglobulin, and the domain utilized in the FLAGS extension/affinity purification system (Immunex, Seattle Wash.). The inclusion of a cleavable-linker sequence such as ENTEROKINASEMAX (Invitrogen, San Diego Calif.) between the protein and the purification domain may also be used to recover the protein.
Suitable host cells may include, but are not limited to, mammalian cells such as Chinese Hamster Ovary (CHO) and human 293 cells, insect cells such as Sf9 cells, plant cells such as Nicotiana tabacum, yeast cells such asSaccharomyces cerevisiae, and bacteria such as E. coli. For each of these cell systems, a useful vector may also include an origin of replication and one or two selectable markers to allow selection in bacteria as well as in a transformed eukaryotic host. Vectors for use in eukaryotic host cells may require the addition of 3xe2x80x2poly(A) tail if the cDNA lacks poly(A).
Additionally, the vector may contain promoters or enhancers that increase gene expression. Many promoters are known and used in the art. Most promoters are host specific and exemplary promoters includes SV40 promoters for CHO cells; T7 promoters for bacterial hosts; viral promoters and enhancers for plant cells; and PGH promoters for yeast. Adenoviral vectors with the rous sarcoma virus enhancer or retroviral vectors with long terminal repeat promoters may be used to drive protein expression in mammalian cell lines. Once homogeneous cultures of recombinant cells are obtained, large quantities of secreted soluble protein may be recovered from the conditioned medium and analyzed using chromatographic methods well known in the art. An alternative method for the production of large amounts of secreted protein involves the transformation of mammalian embryos and the recovery of the recombinant protein from milk produced by transgenic cows, goats, sheep, and the like.
In addition to recombinant production, proteins or portions thereof may be produced manually, using solid-phase techniques (Stewart et al. (1969) Solid-Phase Peptide Synthesis, W H Freeman, San Francisco Calif.; Merrifield (1963) J Am Chem Soc 5:2149-2154), or using machines such as the ABI 431A peptide synthesizer (Applied Biosystems, Foster City Calif.). Proteins produced by any of the above methods may be used as pharmaceutical compositions to treat disorders associated with null or inadequate expression of the genomic sequence.
Screening and Purification Assays
A protein or a portion thereof encoded by the cDNA may be used to screen a library or a plurality of molecules or compounds for a ligand with specific binding affinity or to purify a molecule or compound from a sample. The protein or portion thereof employed in such screening may be free in solution, affixed to an abiotic or biotic substrate, or located intracellularly. For example, viable or fixed prokaryotic host cells that are stably transformed with recombinant nucleic acids that have expressed and positioned a protein on their cell surface can be used in screening assays. The cells are screened against a library or a plurality of ligands and the specificity of binding or formation of complexes between the expressed protein and the ligand may be measured. The ligands may be DNA, RNA, or PNA molecules, agonists, antagonists, antibodies, immunoglobulins, inhibitors, peptides, pharmaceutical agents, proteins, drugs, or any other test molecule or compound that specifically binds the protein. An exemplary assay involves combining the mammalian protein or a portion thereof with the molecules or compounds under conditions that allow specific binding and detecting the bound protein to identify at least one ligand that specifically binds the protein.
This invention also contemplates the use of competitive drug screening assays in which neutralizing antibodies capable of binding the protein specifically compete with a test compound capable of binding to the protein or oligopeptide or fragment thereof. One method for high throughput screening using very small assay volumes and very small amounts of test compound is described in U.S. Pat. No. 5,876,946. Molecules or compounds identified by screening may be used in a model system to evaluate their toxicity, diagnostic, or therapeutic potential.
The protein may be used to purify a ligand from a sample. A method for using a protein to purify a ligand would involve combining the protein or a portion thereof with a sample under conditions to allow specific binding, recovering the bound protein, and using an appropriate chaotropic agent to separate the protein from the purified ligand.
Production of Antibodies
A protein encoded by a cDNA of the invention may be used to produce specific antibodies. Antibodies may be produced using an oligopeptide or a portion of the protein with inherent immunological activity. Methods for producing antibodies include: 1) injecting an animal, usually goats, rabbits, or mice, with the protein, or an antigenically-effective portion or an oligopeptide thereof, to induce an immune response; 2) engineering hybridomas to produce monoclonal antibodies; 3) inducing in vivo production in the lymphocyte population; or 4) screening libraries of recombinant immunoglobulins. Recombinant immunoglobulins may be produced as taught in U.S. Pat. No. 4,816,567.
Antibodies produced using the proteins of the invention are useful for the diagnosis of prepathologic disorders as well as the diagnosis of chronic or acute diseases characterized by abnormalities in the expression, amount, or distribution of the protein. A variety of protocols for competitive binding or immunoradiometric assays using either polyclonal or monoclonal antibodies specific for proteins are well known in the art. Immunoassays typically involve the formation of complexes between a protein and its specific binding molecule or compound and the measurement of complex formation. Immunoassays may employ a two-site, monoclonal-based assay that utilizes monoclonal antibodies reactive to two noninterfering epitopes on a specific protein or a competitive binding assay (Pound (1998) Immunochemical Protocols, Humana Press, Totowa N.J.).
Immunoassay procedures may be used to quantify expression of the protein in cell cultures, in subjects with a particular disorder or in model animal systems under various conditions. Increased or decreased production of proteins as monitored by immunoassay may contribute to knowledge of the cellular activities associated with developmental pathways, engineered conditions or diseases, or treatment efficacy. The quantity of a given protein in a given tissue may be determined by performing immunoassays on freeze-thawed detergent extracts of biological samples and comparing the slope of the binding curves to binding curves generated by purified protein.
Labeling of Molecules for Assay
A wide variety of reporter molecules and conjugation techniques are known by those skilled in the art and may be used in various cDNA, polynucleotide, protein, peptide or antibody assays. Synthesis of labeled molecules may be achieved using commercial kits for incorporation of a labeled nucleotide such as 32P-dCTP, Cy3-dCTP or Cy5-dCTP or amino acid such as 35S-methionine. Polynucleotides, cDNAs, proteins, or antibodies may be directly labeled with a reporter molecule by chemical conjugation to amines, thiols and other groups present in the molecules using reagents such as BIODIPY or FITC (Molecular Probes, Eugene Oreg.).
The proteins and antibodies may be labeled for purposes of assay by joining them, either covalently or noncovalently, with a reporter molecule that provides for a detectable signal. A wide variety of labels and conjugation techniques are known and have been reported in the scientific and patent literature including, but not limited to U.S. Pat. Nos. 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149; and 4,366,241.
Diagnostics
The cDNAs, or fragments thereof, may be used to detect and quantify differential gene expression; absence, presence, or excess expression of mRNAs; or to monitor mRNA levels during therapeutic intervention. Disorders associated with altered expression include liver disorders associated with steroid therapy such as adenomatosis, cholestasis, cirrhosis, hemangioma, Henoch-Schonlein purpura, hepatitis, hepatocellular and metastatic carcinomas, idiopathic thrombocytopenic purpura, porphyria, sarcoidosis, and Wilson disease, and toxicological responses to treatment with steroids and steroid antagonists. These cDNAs can also be utilized as markers of treatment efficacy against the disorders noted above and other disorders, conditions, and diseases over a period ranging from several days to months. The diagnostic assay may use hybridization or amplification technology to compare gene expression in a biological sample from a patient to standard samples in order to detect altered gene expression. Qualitative or quantitative methods for this comparison are well known in the art.
For example, the cDNA may be labeled by standard methods and added to a biological sample from a patient under conditions for hybridization complex formation. After an incubation period, the sample is washed and the amount of label (or signal) associated with hybridization complexes is quantified and compared with a standard value. If the amount of label in the patient sample is significantly altered in comparison to the standard value, then the presence of the associated condition, disease or disorder is indicated.
In order to provide a basis for the diagnosis of a condition, disease or disorder associated with gene expression, a normal or standard expression profile is established. This may be accomplished by combining a biological sample taken from normal subjects, either animal or human, with a probe under conditions for hybridization or amplification. Standard hybridization may be quantified by comparing the values obtained using normal subjects with values from an experiment in which a known amount of a substantially purified target sequence is used. Standard values obtained in this manner may be compared with values obtained from samples from patients who are symptomatic for a particular condition, disease, or disorder. Deviation from standard values toward those associated with a particular condition is used to diagnose that condition.
Such assays may also be used to evaluate the efficacy of a particular therapeutic treatment regimen in animal studies and in clinical trial or to monitor the treatment of an individual patient. Once the presence of a condition is established and a treatment protocol is initiated, diagnostic assays may be repeated on a regular basis to determine if the level of expression in the patient begins to approximate that which is observed in a normal subject. The results obtained from successive assays may be used to show the efficacy of treatment over a period ranging from several days to months.
Gene Expression Profiles
A gene expression profile comprises a plurality of cDNAs and a plurality of detectable hybridization complexes, wherein each complex is formed by hybridization of one or more probes to one or more complementary sequences in a sample. The cDNAs of the invention are used as elements on a microarray to analyze gene expression profiles. In one embodiment, the microarray is used to monitor the progression of disease. Researchers can assess and catalog the differences in gene expression between healthy and diseased tissues or cells. By analyzing changes in patterns of gene expression, disease can be diagnosed at earlier stages before the patient is symptomatic. The invention can be used to formulate a prognosis and to design a treatment regimen. The invention can also be used to monitor the efficacy of treatment. For treatments with known side effects, the microarray is employed to improve the treatment regimen. A dosage is established that causes a change in genetic expression patterns indicative of successful treatment. Expression patterns associated with the onset of undesirable side effects are avoided. This approach may be more sensitive and rapid than waiting for the patient to show inadequate improvement, or to manifest side effects, before altering the course of treatment.
In another embodiment, animal models which mimic a human disease can be used to characterize expression profiles associated with a particular condition, disorder or disease; or treatment of the condition, disorder or disease. Novel treatment regimens may be tested in these animal models using microarrays to establish and then follow expression profiles over time. In addition, microarrays may be used with cell cultures or tissues removed from animal models to rapidly screen large numbers of candidate drug molecules, looking for ones that produce an expression profile similar to those of known therapeutic drugs, with the expectation that molecules with the same expression profile will likely have similar therapeutic effects. Thus, the invention provides the means to rapidly determine the molecular mode of action of a drug.
Assays Using Antibodies
Antibodies directed against epitopes on a protein encoded by a cDNA of the invention may be used in assays to quantify the amount of protein found in a particular human cell. Such assays include methods utilizing the antibody and a label to detect expression level under normal or disease conditions. The antibodies may be used with or without modification, and labeled by joining them, either covalently or noncovalently, with a labeling moiety.
Protocols for detecting and measuring protein expression using either polyclonal or monoclonal antibodies are well known in the art. Examples include ELISA, RIA, and fluorescent activated cell sorting (FACS). Such immunoassays typically involve the formation of complexes between the protein and its specific antibody and the measurement of such complexes. These and other assays are described in Pound (supra). The method may employ a two-site, monoclonal-based immunoassay utilizing monoclonal antibodies reactive to two non-interfering epitopes, or a competitive binding assay. (See, e.g., Coligan et al. (1997) Current Protocols in Immunology, Wiley-Interscience, New York N.Y.; Pound, supra)
Therapeutics
The cDNAs and fragments thereof can be used in gene therapy. cDNAs can be delivered ex vivo to target cells, such as cells of bone marrow. Once stable integration and transcription and or translation are confirmed, the bone marrow may be reintroduced into the subject. Expression of the protein encoded by the cDNA may correct a disorder associated with mutation of a normal sequence, reduction or loss of an endogenous target protein, or overepression of an endogenous or mutant protein. Alternatively, cDNAs may be delivered in vivo using vectors such as retrovirus, adenovirus, adeno-associated virus, herpes simplex virus, and bacterial plasmids. Non-viral methods of gene delivery include cationic liposomes, polylysine conjugates, artificial viral envelopes, and direct injection of DNA (Anderson (1998) Nature 392:25-30; Dachs et al. (1997) Oncol Res 9:313-325; Chu et al. (1998) J Mol Med 76(3-4):184-192; Weiss et al. (1999) Cell Mol Life Sci 55(3):334-358; Agrawal (1996) Antisense Therapeutics, Humana Press, Totowa N.J.; and August et al. (1997) Gene Therapy (Advances in Pharmacology, Vol. 40), Academic Press, San Diego Calif.).
In addition, expression of a particular protein can be regulated through the specific binding of a fragment of a cDNA to a genomic sequence or an mRNA which encodes the protein or directs its transcription or translation. The cDNA can be modified or derivatized to any RNA-like or DNA-like material including peptide nucleic acids, branched nucleic acids, and the like. These sequences can be produced biologically by transforming an appropriate host cell with a vector containing the sequence of interest.
Molecules which regulate the activity of the cDNA or encoded protein are useful as therapeutics for liver disorders associated with steroid therapy such as adenomatosis, cholestasis, cirrhosis, hemangioma, Henoch-Schonlein purpura, hepatitis, hepatocellular and metastatic carcinomas, idiopathic thrombocytopenic purpura, porphyria, sarcoidosis, and Wilson disease, and for regulating metabolic and toxicological responses to treatment with steroids and steroid antagonists. Such molecules include agonists which increase the expression or activity of the polynucleotide or encoded protein, respectively; or antagonists which decrease expression or activity of the polynucleotide or encoded protein, respectively. In one aspect, an antibody which specifically binds the protein may be used directly as an antagonist or indirectly as a delivery mechanism for bringing a pharmaceutical agent to cells or tissues which express the protein.
Additionally, any of the proteins, or their ligands, or complementary nucleic acid sequences may be administered as pharmaceutical compositions or in combination with other appropriate therapeutic agents. Selection of the appropriate agents for use in combination therapy may be made by one of ordinary skill in the art, according to conventional pharmaceutical principles. The combination of therapeutic agents may act synergistically to affect the treatment or prevention of the conditions and disorders associated with an immune response. Using this approach, one may be able to achieve therapeutic efficacy with lower dosages of each agent, thus reducing the potential for adverse side effects. Further, the therapeutic agents may be combined with pharmaceutically-acceptable carriers including excipients and auxiliaries which facilitate processing of the active compounds into preparations which can be used pharmaceutically. Further details on techniques for formulation and administration used by doctors and pharmacists may be found in the latest edition of Remington""s Pharmaceutical Sciences (Mack Publishing, Easton Pa.).
Model Systems
Animal models may be used as bioassays where they exhibit a phenotypic response similar to that of humans and where exposure conditions are relevant to human exposures. Mammals are the most common models, and most infectious agent, cancer, drug, and toxicity studies are performed on rodents such as rats or mice because of low cost, availability, lifespan, reproductive potential, and abundant reference literature. Inbred and outbred rodent strains provide a convenient model for investigation of the physiological consequences of underexpression or overexpression of genes of interest and for the development of methods for diagnosis and treatment of diseases. A mammal inbred to overexpress a particular gene (for example, secreted in milk) may also serve as a convenient source of the protein expressed by that gene.
Transgenic Animal Models
Transgenic rodents that overexpress or underexpress a gene of interest may be inbred and used to model human diseases or to test therapeutic or toxic agents. (See, e.g., U.S. Pat. Nos. 5,175,383 and 5,767,337.) In some cases, the introduced gene may be activated at a specific time in a specific tissue type during fetal or postnatal development. Expression of the transgene is monitored by analysis of phenotype, of tissue-specific mRNA expression, or of serum and tissue protein levels in transgenic animals before, during, and after challenge with experimental drug therapies.
Embryonic Stem Cells
Embryonic (ES) stem cells isolated from rodent embryos retain the potential to form embryonic tissues. When ES cells such as the mouse 129/SvJ cell line are placed in a blastocyst from the C57BL/6 mouse strain, they resume normal development and contribute to tissues of the live-born animal. ES cells are preferred for use in the creation of experimental knockout and knockin animals. The method for this process is well known in the art and the steps are: the cDNA is introduced into a vector, the vector is transformed into ES cells, transformed cells are identified and microinjected into mouse cell blastocysts, blastocysts are surgically transferred to pseudopregnant dams. The resulting chimeric progeny are genotyped and bred to produce heterozygous or homozygous strains.
Knockout Analysis
In gene knockout analysis, a region of a gene is enzymatically modified to include a non-natural intervening sequence such as the neomycin phosphotransferase gene (neo; Capecchi (1989) Science 244:1288-1292). The modified gene is transformed into cultured ES cells and integrates into the endogenous genome by homologous recombination. The inserted sequence disrupts transcription and translation of the endogenous gene.
Knockin Analysis
ES cells can be used to create knockin humanized animals or transgenic animal models of human diseases. With knockin technology, a region of a human gene is injected into animal ES cells, and the human sequence integrates into the animal cell genome. Transgenic progeny or inbred lines are studied and treated with potential pharmaceutical agents to obtain information on the progression and treatment of the analogous human condition.
As described herein, the uses of the cDNAs, provided in the Sequence Listing of this application, and their encoded proteins are exemplary of known techniques and are not intended to reflect any limitation on their use in any technique that would be known to the person of average skill in the art. Furthermore, the cDNAs provided in this application may be used in molecular biology techniques that have not yet been developed, provided the new techniques rely on properties of nucleotide sequences that are currently known to the person of ordinary skill in the art, e.g., the triplet genetic code, specific base pair interactions, and the like. Likewise, reference to a method may include combining more than one method for obtaining or assembling full length cDNA sequences that will be known to those skilled in the art. It is also to be understood that this invention is not limited to the particular methodology, protocols, and reagents described, as these may vary. It is also understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention which will be limited only by the appended claims. The examples below are provided to illustrate the subject invention and are not included for the purpose of limiting the invention.